Method to prepare dye-based reference material

ABSTRACT

Provided is a method of preparing dye-based reference materials useful for calibrating or qualifying instrument systems that are diagnostic spectroscopically for hemoglobin and CO-ox fractions. The dye-based reference materials are non-proteinaceous and may be interpreted by CO-oximeter instrument systems as providing a spectrum substantially equivalent to the spectrum of blood, particularly with regard to hemoglobin and hemoglobin CO-ox fractions. Dye-based reference materials may be formulated to provide CO-ox fractions approximating those seen in a clinical setting, including normal physiological CO-ox fractions. The dye-based reference materials may be combined with other ingredients thus making the materials useful as references for pH and blood gas instruments and/or electrolyte instruments in addition to CO-oximeter instrument systems.

This is a continuation of application Ser. No. 08/447,168, filed on May19, 1985, now U.S. Pat. No. 5,637,505, issued Jun. 10, 1997.

FIELD OF INVENTION

This invention relates to a method of preparing dye-based referencematerials useful for calibrating or qualifying instrument systems thatare diagnostic spectroscopically for hemoglobin and CO-ox fractions. Thereference materials may also be used in instrument systems that have, inaddition to capability to measure CO-ox fractions, sensors for themeasurement of blood pH, gas and other blood analytes includingelectrolyte concentrations and metabolite concentrations.

BACKGROUND OF THE INVENTION

Improvements in instrumentation have made the determination of blood pH,gas, electrolytes and CO-ox fractions relatively routine in clinicallaboratories. Typically, pH and blood gas instruments measure blood pH,pCO₂, and pO₂. CO-oximeter instruments typically measure the totalhemoglobin concentration (THb); the hemoglobin fractions such asoxyhemoglobin (O₂ Hb); methemoglobin (MetHb); carboxyhemoglobin (COHb);and reduced hemoglobin (HHb) (collectively referred to herein as "CO-oxfractions"). Electrolyte instruments measure any number of floodelectrolytes, including sodium, potassium, lithium, calcium, and so on.Instrument systems currently available may combine the measurement ofblood pH, gases, electrolytes, various metabolites, and CO-ox fractionsin one instrument for a comprehensive testing of the properties ofblood, as is particularly useful in respiratory and pulmonary ailments.Vigorous therapeutic treatment is often dictated by such test results.

Quantitative determination of the various CO-ox fractions in clinicalsettings is desirable as CO-ox fractions relate to the loading of oxygenonto the hemoglobin of red blood cells circulating through the pulmonarycapillaries. The actual amount of oxygen loaded onto the hemoglobin isdetermined not only by the concentration of total hemoglobin (THb), butalso by the amount of non-oxygen-binding derivatives of hemoglobin suchas carboxyhemoglobin (COHb) and methemoglobin (MetHb). Reducedhemoglobin (HHb) is an unoxygenated form of normal hemoglobin andelevations in the arterial fractional HHb indicate that lesser amountsof oxygen have been bound as a result of ventilation and/or perfusiondefects.

Blood, hemoglobin and hemoglobin CO-ox fractions absorb visible light. Anormal blood spectrum has a main absorption peak at 578 nm and decreasesrapidly close to zero at wavelengths greater than about 610 nm as shownin FIG. 1. The second absorption peak of blood is at 542 nm. Absorbancemaxima of hemoglobin derivatives are oxyhemoglobin, 541, 568-572 nm;reduced hemoglobin, 555 nm; carboxyhemoglobin, 537, 568-572 nm; andmethemoglobin 540, 578, 630 nm.

Generally, optical type of CO-oximeters measure the absorbance of theblood sample at multiple wavelengths on the spectrum. Ultimately, basedon the known CO-ox fraction absorption wavelengths regions, CO-oximetersanalyze blood samples by the collection of absorption data at specificwavelengths. The data are then typically recorded and a process calledmulticomponent analysis is used to simultaneously calculate theconcentrations of the each of hemoglobin CO-ox fraction present in theblood sample.

CO-oximeter instruments are typically designed to measure CO-oxfractions with values broader than the ranges observed in patientsamples. For example, instruments may have the capability to measureCO-ox fractions in the defined instrument ranges of: about 4 to about 25g/dL of THb, about 30% to about 98% oxyhemoglobin (O₂ Hb); 0 to about50% carboxyhemoglobin (COHb); 0 to about 40% methemoglobin (MetHb); and0 to about 50% reduced hemoglobin (HHb), with all CO-ox fractionpercentages herein based on the total amount of hemoglobin. Within therange of instrument capability is a clinically meaningful CO-ox fractionrange and a normal physiological CO-ox fraction range. The clinicallymeaningful range is defined herein as: total hemoglobin 8 to 20 g/dL, 60to 98% oxyhemoglobin (O₂ Hb); 0 to 20% carboxyhemoglobin (COHb); 0 toabout 20% methemoglobin (MetHb); and 0 to about 20% reduced hemoglobin(HHb). The normal physiological range is defined herein as: totalhemoglobin.14 to 17 g/dL for men and 12 to 15 g/dL for women,oxyhemoglobin 94 to 98%, carboxyhemoglobin 0 to about 1% for nonsmoker,methemoglobin 0 to about 1.5%, and reduced hemoglobin from about 1 toabout 5%.

Reference materials generally function to validate the performance of adiagnostic instrument. For CO-oximeter instrument systems, ideal qualitycontrol standard materials are formulated to provide pre-determinedCO-ox fraction values not only within the broad instrument capabilityrange, but also within the normal physiological range.

The prior art teaches two general types of CO-oximeter quality controlstandard materials. The first type are aqueous dye-based materials,where dyes are used in an attempt to match the spectrum of blood. Thesecond type are blood-based materials, where the presence blood allowsfor the direct match of the spectrum of blood. Both types of materialshave been associated with a variety of problems as discussed below.

In developing dye-based quality control standard materials for CO-oxinstrument systems, to more closely approximate the spectrum of blood,combinations of dyes have been used. The combinations of dyes have beenused because no single synthetic dye has a spectrum sufficiently similarto the absorption bands of a blood spectrum. Because the spectrum ofblood has multiple distinctive bands of absorption, it is challenging toprepare a quality control standard material to mimic thesecharacteristics. Although one dye might contribute an absorptioncharacteristic that is present in the normal blood spectrum, it may alsopresent other absorption characteristics that are dissimilar to blood atother portions of the spectrum.

Prior art methods teach dye combinations that only partially simulateblood's visible spectrum. Consequently, the CO-ox fraction values of theprior art quality control standard materials are often not clinicallymeaningful. For example, U.S. Pat. No. 4,843,013 teaches a combinationof dyes for a CO-ox quality control standard, however, as shown in Cols.6 and 7 of U.S. Pat. No. 4,843,013, the described quality controlstandard provides negative values for some of the CO-ox fractions. Anegative value for a CO-ox fraction would never appear in a blood sampleand thus these control standards have limited usefulness in qualifyingCO-oximeters in a clinical setting. There has been a long-feltcommercial need for dye-based quality control materials that may beformulated in a predictable manner to provide pre-determined clinicallymeaningful CO-ox fractions, particularly in the normal physiologicalrange.

Blood-based reference materials provide clinically meaningful CO-oxfraction values, however, numerous limitations have been associated withusing blood in the quality control standard materials. For example, asignificant problem encountered by users of the proteinaceousblood-based materials is that blood-based materials are very susceptibleto bacterial contamination and have a limited shelf life. Consequently,in most cases, users of blood-based materials must refrigerate theproducts during storage. Additionally, blood-based reference materialsare generally classified as biohazardous materials, thus requiring theuser to take additional safety precautions.

There is a need to provide non-proteinaceous dye-based referencematerials that provide clinically and physiologically meaningful CO-oxfraction ranges.

SUMMARY OF THE INVENTION

According to the invention, a method to prepare a reference material forCO-oximeter instrument systems is provided, said method comprising (a)selecting a red dye having a main absorption maxima at a wavelengthregion between about 560 to about 580 nm to provide a basic spectrum;(b) modifying said main absorption maxima of said red dye by using a dyehaving an absorption maxima at a wavelength region between about 350 toabout 450 nm; and (c) establishing a new relative baseline of saidspectrum at wavelengths greater than about 640 nm and providing asufficient absorbance at about 630 nm to provide a positivemethemoglobin CO-ox fraction using one or more dyes.

Further provided is a reference material prepared according to saidmethod, wherein said dyes are used in sufficient amounts to provide areference material with CO-ox fractions falling within a range coveringinstrument capability. The method may also be used to prepare referencematerials having CO-ox fractions with values falling within the definedclinical range or within the defined normal physiological range.

Also provided in one embodiment is a reference material comprising (a) afirst dye having a main absorption maxima at a wavelength between about560 to about 580 nm; (b) a second dye having an absorption maxima at awavelength between about 350 to about 450 nm; and (c) a third dye havingan absorption maxima at a wavelength between about 625 and about 640 nm;and (d) a fourth dye having an absorption maxima at a wavelength betweenabout 700 to about 780 nm and an absorption band width of from about 150to about 200 nm. The amounts of the dyes used in the materials may beadjusted to provide quality control materials with desired CO-oxfraction values.

Further provided is a method for quality control of CO-oximeterinstrument systems comprising: (1) subjecting said instrument to aquality control standard material comprising (a) a first dye having amain absorption maxima at a wavelength between about 560 to about 580nm; (b) a second dye having an absorption maxima at a wavelength betweenabout 350 to about 450 nm; and (c), a third dye having an absorptionmaxima at a wavelength between about 625 and about 640 nm; and (d) afourth dye-having an absorption maxima at a wavelength between about 700to about 780 nm and an absorption band width of from about 150 to about200 nm; wherein said dyes are used in a sufficient amount to providesaid control with a pre-determined level of CO-ox fractions; (2)obtaining an instrument measurement of CO-ox fractions of said control;and (3) comparing said instrument measurement of said CO-ox fractions ofsaid control with said predetermined level of CO-ox fractions to checkthe accuracy of said instrument.

The dye-based reference materials are non-proteinaceous and may beinterpreted by CO-oximeter instrument systems as providing a spectrumsubstantially equivalent to the spectrum of blood, particularly withregard to total hemoglobin and hemoglobin CO-ox fractions. Using theinventive method of preparing the reference materials, dye-basedreference materials may be formulated to provide CO-ox fractionsapproximating those seen in a clinical setting, including normalphysiological values. These dye-based reference materials may also becombined with other ingredients which make the materials useful asreference materials for pH and blood gas instruments and/or electrolyteinstruments in addition to CO-oximeter instruments.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the spectrum of blood.

FIG. 2 shows the spectrums of single dyes and a dye combination thatdoes not provide clinically meaningful CO-ox fractions. Spectrum 1 istaken from a sulforhodamine B solution. Spectrum 2 is taken from asulforhodamine 101 solution. Spectrum 3 is taken from a solutioncontaining 1 g/L sulforhodamine B and 0.1 g/L sulforhodamine 101.

FIG. 3 illustrates spectroscopically how the spectrum of sulforhodamineB is modified using the inventive method of formulation. "SRB" is anabbreviation for sulforhodamine B; "MY7" is an abbreviation for mordantyellow 7; "PBV" is an abbreviation for patent blue violet; and "NGB" isan abbreviation for naphthol green B.

FIGS. 4-7 show,the spectra of the three levels of formulations shown inExamples 1-4, respectively.

DETAILED DESCRIPTION OF INVENTION

The present invention provides a method to formulate dye-based referencematerials which mimic the spectrum of human blood when the referencematerials are utilized in optical types of CO-oximeter instrumentsystems. The formulating method is described herein in distinct stepsfor purposes of clarity only. It should be understood that the steps setforth may be carried out in any order and may also be combined andcarried out simultaneously. Additionally, the designation of first dye,second dye, and so on, is not intended to imply a specific order ofdyes. The types of dyes selected, the amount of dyes utilized and theconcentration of dyes described in these steps may be adjusted until thedesired level of hemoglobin CO-ox fractions are obtained, as preferablydetermined by taking a reading from the specific CO-oximeter instrumentthat will be used with the reference material. All measurements of CO-oxfractions set forth herein were taken on Ciba Corning M270 CO-oximeters(as available from Ciba Corning Diagnostics Corp., Medfield, Mass. USA).All measurements of blood gas and electrolytes set forth herein weretaken on Ciba Corning M288 Blood Gas Analyzers.

According to the invention, in step (a) a red dye is selected to providea basic spectrum (also referred to herein as the first dye). Inselecting the red dye, the general characteristics of the spectrum ofnormal blood should be considered. Normal blood has a spectrum with amain absorption peak at 578 nm which decreases rapidly close to zero ata wavelength greater than about 610 nm as shown in FIG. 1. The secondabsorption peak of blood is at approximately 542 nm. The absorbancemaxima of the CO-ox fraction oxyhemoglobin is at the approximatewavelengths of 541 nm and 568-572 nm. As normal arterial blood has anoxyhemoglobin fraction greater than about 95%, the dye-based formulationshould have a red dye with a spectrum similar to at least the generalabsorption features of oxyhemoglobin. The preferred red dyes in theinvention have an absorption peak in the wavelength range from about 560nm to about 580 nm. Further, particularly preferred red dyes exhibitminimal absorbance at a wavelength of greater than about 610 nm. Reddyes, such as, for example, sulforhodamine B, chlorophenol red, xylenolorange, sulforhodamine 101, etc., are currently preferred. Mostpreferably employed as the red dye is sulforhodamine B, which has anabsorption maximum at approximately 566 nm in aqueous solution, a fairlynarrow absorption bandwidth, and an absorbance which decreases to zeroat about 620 nm, as shown as spectrum 1 in FIG. 2.

The single red dye does not exhibit a spectrum sufficiently similar tothat of blood for purposes of obtaining clinically meaningful CO-oxfractions, as exemplified by the CO-ox fractions measured from 1.2 g/Lof sulforhodamine B: THb 14 g/dL, O₂ Hb -16%, COHb 105%, MetHb -2.5% andHHb 13.5%. As these values indicate, negative CO-ox fractions areobtained for the oxyhemoglobin and the methemoglobin.

One approach to alter the absorption peak of the red dye might be tointroduce another red dye. In this approach, to shift the absorptionpeak to a longer wavelength, another red dye with absorption maximumwavelength greater than that of the first red dye could be utilized.Alternatively, to shift absorption peak to a shorter wavelength, anotherred dye with absorption maximum wavelength less than that of the firstred dye could be utilized. A combination of the two red dyes would bepredicted to result in a new overlapped maximum that would complementthe required absorption features. However, as shown in FIG. 2,sulforhodamine B in aqueous solution provides a spectrum that does nothave enough strength of absorbance at 570 to 585 nm because theabsorption peak of the sulforhodamine B is located at a shorterwavelength than that of blood. Sulforhodamine 101 has an absorption peakat 587 nm. A combination of sulforhodamine B and sulforhodamine 101shifts the absorption peak of sulforhodamine B and enhances theabsorbance at 570 nm to 585 nm. In FIG. 2, spectrum 1 is sulforhodamineB. spectrum 2 is sulforhodamine 101, and spectrum 3 is the combinationof 1 g/L sulforhodamine B and 0.1 g/L sulforhodamine 101. As shown inFIG. 2, absorbance of the two red dye combination at 570 to 585 nm isenhanced and results in the change of CO-ox fractions from negativevalues to positive values, e.g., 14 g/dL THb, 16% O₂ Hb, 1% MetHb, 14%COHb and 69% HHb. The addition of the second red dye (sulforhodamine101) introduces an undesired high absorbance near wavelength range of590 nm to 605 nm, thus not providing an acceptable method of formulatingreferences having clinically meaningful and/or normal physiologicalCo-ox fraction values.

In step (b), modification and manipulation of the absorption band of thered dye is conducted where the absorption peak of the red dye is shiftedto a longer wavelength to more closely approximate that of blood. Uponstep (b), the absorption maxima of the red dye is shifted to fallbetween the wavelengths of approximately 570 nm to about 585 nm. It ispreferred that the shift of the absorption maxima does not inadvertentlyincrease the absorption in the wavelength region of 590 to 605 nm. Ithas been found that dye(s) having an absorption maxima at wavelengths ofabout 350 to 450 nm are useful in step (b) because the dye(s) increasethe absorbance of the spectrum at the ultraviolet region and force theabsorption band of the red dye to shift toward the longer wavelength.

Preferably in step (b) is accomplished using a yellow dye (or acombination thereof) including, for example, mordant yellow 7,tartrazine, orange G, hydroxypyrenetrisulfonic acid, mordant yellow 10,combinations thereof, and so on. Particularly preferred is mordantyellow 7 dye (especially when sulforhodamine B is. selected as the reddye).

FIG. 3 illustrates the changes of the spectrum of sulforhodamine B afteraddition of mordant yellow 7. Spectrum 1 in FIG. 3 is the sulforhodamineB aqueous solution, and spectrum 2 is the sulforhodamine B aqueoussolution containing mordant yellow 7. As shown, the original absorptionpeak of sulforhodamine B is shifted from 566 nm to 572 nm, with the edgeof the absorption band at approximately 610 nm, and with little increaseof absorbance in the wavelength range of 590 to about 605 nm.

According to the present invention, in step (c) a new relative baselineof the spectrum is established at wavelengths greater than about 640 nmand sufficient absorbance at about 630 nm is provided to yield apositive methemoglobin CO-ox fraction. Step (c) addresses that theabsorbance at about 590 to about 605 nm of the dye or dye combinationused in steps (a) and (b) is higher than that of normal blood. This stepmay be accomplished using one or more dyes. With respect to the newlifted baseline, the original absorbance can be normalized, with thenormalized absorbance reduced and smaller than the original absorbance.Further, the original absorbance at about 590 to about 605 nm, althoughhaving a similar absolute absorbance value, is relatively reduced uponthe establishment of the new lifted baseline. CO-oximeters take thelifted baseline from the CO-oximeters interpret the dye spectrum, andcalculate the CO-ox fractions therefrom. Upon step (c), the inventivereference material will read as providing CO-ox fraction values withinthe instrument capability and clinically meaningful ranges, andoptionally, within the normal physiological range, as described in moredetail hereinafter.

One group of dyes particularly useful in step (c) are designated hereinas the third dye(s). These dyes have an absorption maxima at about 625to about 640 nm (more preferably about 630 nm) and have only minimumimpact on the absorbance at wavelengths shorter than about 600 nm.Preferably, the third dye is a blue dye selected from brilliant blue FC(FD&C blue 1), erioglaucine, lissamine, alphazurine A, patent blueviolet, hydroxy naphthol blue, patent blue VF, combinations thereof, andequivalents thereof (with patent blue violet, patent blue VF, andalphazurine more preferred). Patent blue violet has an absorption maximaat about 639 nm and is most preferred because it provides the absorbancenot only at 630 nm, but also absorbance to some extent at a wavelengthgreater than 640 nm, thus generally accomplishing both functions of step(c). Adding the patent blue violet into the sulforhodamine B and yellowmordant 7 combination pushes the CO-ox fractions to the following: O₂ Hb80%, MetHb 15%, COHb 2% and HHb 3%, thus placing the reference materialwithin the values set forth for instrument capability and clinicallymeaningful CO-ox fractions.

A second group of dyes that may be used in step (c) are designated asthe fourth dye(s). These dyes have an main absorbance maxima positionedat a wavelength of from about 700 to about 780 nm and an absorption bandwidth of from about 150 to about 200 nm. The tail of the absorption bandof the dye (or dyes) of step (c) preferably lifts the baseline of theabsorbance spectrum with minimal effect on absorbance at 500 to 605 nm.Preferred dyes falling within this description may be selected from anynumber of dyes including IR 125, naphthol green B, and combinationsthereof, and so on. Most preferred is naphthol green B, having anabsorption maxima at approximately 716 nm in an aqueous solution, andconsidered particularly effective when combined with mordant yellow 7,patent violet blue and sulforhodamine B.

For step (c) the combination of the third and fourth dyes allow thereference material containing the first and second dyes to yield CO-oxfraction values falling with the normal, physiological range. FIG. 3exemplifies the principles of the present method of formulation. Thesulforhodamine B (SRB) is the red dye that provides a basic spectrum.The addition of mordant yellow 7 (MY7) to the sulforhodamine B (SRB)modifies the spectrum by tilting the absorption band of thesulforhodamine B towards a longer wavelength and shifts the absorptionmaxima from about 566 nm to about 572 nm (SRB+MY7), to more closelyapproximate the main absorption spectrum of blood. Patent blue violet(PBV) is added for purposes of providing a spectrum absorption formethemoglobin at approximately 630 nm (SRB+MY7+PBV) and also slightlylifts the baseline spectrum. The addition of the naphthol green B (NGB)further lifts the spectrum baseline to the right of the main absorptionmaxima. The ultimate result is shown by Spectrum "SRB+MY7+PBV+NGB". Inthis formulation the following amounts of dyes were used: about 1.15 g/Lsulforhodamine B; about 2.4 g/L mordant yellow 7; about 0.08 g/L patentblue violet; and about 1 g/L naphthol green B. When this combination wasmeasured on a Ciba Corning M270 CO-oximeter the following CO-ox fractionranges (which are within the normal physiological range) were provided:THb 14 g/dL, O₂ Hb 96%, COHb 1%, MetHb 0.8% and HHb 2.2%. Providing areference material having an oxyhemoglobin value greater than about 60%has been met with limited success in the past, however, the presentinvention provides a method to easily yield this objective.

Any suitable dyes may be used in the formulation of the referencematerials with either aqueous or nonaqueous solvents. Water soluble dyesare particularly well-suited, including acid dyes, basic dyes, directdyes, and so on, and equivalents thereof. The dye composition may beprepared as a dry material for ease of storage and packaging. Ifprepared as a dry composition, prior to usage the composition may beprepared as a solution using a suitable liquid, including water andvarious organic solvents, or mixtures thereof and so on, by techniqueswell known to those skilled in the art. It is particularly preferredthat compatible dyes are used, with a particularly preferred embodimentutilizing anionic dyes. Although the method of formulating may beaccomplished using various amounts of dyes, a particularly preferredcomposition employs a total dye concentration of from about 0.1 to about10 mM. Further, the stability of the reference materials will beincreased when high purity dyes, which are either commercially availableor purified, using conventional methods known to those skilled in theart, are used in formulation.

The reference materials prepared according to the invention may beutilized on various types of optical CO-oximeters (or instrumentsincorporating a CO-oximeter function). Because various types ofCO-oximeters may be designed and constructed to measure the absorbanceof blood at different set of wavelengths, the reference materials usedfor these CO-oximeters will typically need dye concentration adjustmentfor this variation, where the adjustment may be accomplished by thoseskilled in the art employing the principles of this invention.

The reference materials may be prepared as calibration materials tosimultaneously calibrate instrument systems that include sensors forblood gas, electrolyte, and various metabolites in addition to theCO-oximeter capability. Further, the reference materials areparticularly well-suited for preparation as quality control materialsfor instrument systems that include a CO-oximeter capability, inaddition to various other sensors, as described herein. For CO-oximeterinstrument systems, ideally, the CO-0x fraction accuracy is generallychecked both in the broader instrument capability CO-ox fraction rangeas well as in the more narrow normal physiological range (as definedherein). More preferably, the CO-oximeter instrument system is checkedfor performance accuracy on at least three levels. These three referencematerials are preferably formulated to provide pre-determined CO-oxfraction values for the total hemoglobin as well as various hemoglobinCO-ox fractions outside of and within normal physiological ranges.

Particularly preferred compositions provide a commercially acceptablestability. The stability may also extend to high temperature stabilitysuch that the composition may be autoclaved for purposes of facilitatingsterilization, if desired. Additionally, the dye formulations may beprepared such that they are stable to exposure to light.

Appropriate ingredients that be included in the dye formulationpreferably are those ingredients that do not interfere with the sensorsof the instruments such that the reference materials may be used toprovide calibration or quality control of different types of sensorssimultaneously. A preferred reference prepared using the dye formulationof this invention may be prepared by combining in the appropriaterelative proportions, buffer(s), dyes, sources of analytes (typicallysalts), and other standard chemicals commonly used in referencematerials including preservatives and surfactants, and so on, as wellknown in the art. Buffer materials that may be used, particularly whenpreparing a reference standard for simultaneous usage on pH blood gasand electrolyte sensor system, are preferably selected for having a pKaclose to the desired working pH. Particularly useful buffer materialsare zwitterion buffers that are compatible with the selected dyes,including, for example, N-2-hyroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES), 3-(N-morpholinio)propanesulfonic acid (MOPS),Tris-(Hydroxymethyl)aminomethane (TRIS), and so on, as are well known tothose skilled in the art. A combination of the salts such as, forexample, NaCl, NaOH, KCl, LiCl, NaHCO₃, Na₂ SO₄, CaCl₂ glucose, lithiumlactate, and equivalents thereof, can be used to provide the desiredconcentration of electrolytes, as is well known to those skilled in theart. A stable ionized calcium concentration may be provided by usingcalcium chelating agents, such as, ethylenediaminetetraacetic acid(EDTA), N-hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid(NTA), and citric acid. Further, when providing a reference for a bloodgas measurement, the desired pCO₂ and pO₂ ranges may be reached bytonometering to an equilibrium at approximately 25° C. using gascombination with proper oxygen, carbon dioxide, and nitrogen contents,as well within the skill of those trained in the art.

A particularly useful reference material includes the above-describeddyes providing normal physiological Co-ox fractions while in combinationwith an aqueous solution of known quantities of pH, pO₂, pCO₂, Na, K,Cl, and ionized Ca (iCa), glucose, and lactate (preferably in amountsthat typically fall within the range of a blood sample). After saidcombination, the reference material may be divided into aliquots whichare placed in sealed receptacles. Thereafter the receptacles may beflushed with tonometry gas, and the receptacle sealed. These referencesmay be used for verifying the accuracy and reliability of instrumentsystems having multiple sensors for measuring blood pH, gas, CO-oxfractions and various electrolyte and metabolite levels. A particularlypreferred embodiment that is described in TABLE A below.

                  TABLE A    ______________________________________    COMPOUND      APPROXIMATE CONCENTRATIONS    ______________________________________    NaCl          110 to 160 mM    KCl           2.5 to 7.5 mM    CaCl.sub.2    2 to 4 mM    HEPES         35 to 40 mM    TRIS.HCl      15 to 40 mM    NaOH          20 to 50 mM    EDTA          1 to 1.7 mM    NaHCO.sub.3   15 to 20 mM    LiCl          10 to 25 mM    Glucose       0.5 to 10 g/L    Li lactate    0.5 to 14 mM    Sulforhodamine B                  0.55 to 1.3 g/L    Mordant Yellow 7                  1.4 to 3 g/L    Patent Blue Violet                  0.07 to 0.08 g/L    Naphthol Green B                  0.4 to 1.1 g/L    ______________________________________

To prepare clinically meaningful reference materials, preferably thefirst dye is used in an amount ranging from about 0.55 mM to about 4 mM;the second dye is used in an amount ranging from about 1 mM to about 10mM; the third dye is used in an amount ranging from about 0.01 mM to 0.3mM; and the fourth dye is used in an amount ranging from 0 to about 2.5mM.

It is to be understood that various modifications to the invention willbe apparent to and can readily be made by those skilled in the art,given the disclosure herein, without departing from the scope andmaterials of this invention. It is noted that the following examplesgiven herein are intended to illustrate and not to limit the inventionthereto.

EXAMPLES Example 1

A three-level quality control standard material was prepared withvarying levels of pH, pCO₂, pO₂, sodium (Na), potassium (K), chloride(Cl), ionized calcium (iCa), and three levels of total hemoglobin (THb),oxyhemoglobin (O₂ Hb), carboxyhemoglobin (COHb), methemoglobin (MetHb),and reduced hemoglobin (HHb), as shown in TABLES I and II. The threelevels prepared represent clinically significant ranges of acidosis(level a), normal or physiological (level b), and alkalosis (level c).

More particularly, three bulk solutions containing the required saltswere prepared in an aqueous solution, as shown below in TABLE I.

                  TABLE I    ______________________________________    BULK SOLUTIONS    COMPOUND    Level a     Level b   Level c    ______________________________________    NaCl        55.5 mM     60 mM     65 mM    KCl         3.20        4.85      710    CaCl.sub.2  3.81        2.80      2.13    NaOH        29.5        38        34.3    LiCl        21.5        12.9      --    EDTA        1.63        1.20      1.14    HEPES       40          40        40    TRIS.HCl    35.5        15        --    TRIS base   10          --        --    NaHCO.sub.3 19.5        23.4      19.5    ______________________________________

To the bulk solutions described in TABLE I, three levels of dyeformulations were added, as shown in TABLE II.

                  TABLE II    ______________________________________    DYE FORMULATIONS    Dye           Level a   Level b   Level c    ______________________________________    Sulforhodamine B                  0.98 mM   1.95 mM   2.27 mM    Mordant Yellow 7                  6.65      7.63      3.89    Patent Blue Violet                  0.14      0.11      0.14    Naphthol Green B                  --        1.10      0.48    ______________________________________

After dyes dissolved in the bulk solution, the solutions were thentonometered at 25° C. to an equilibrium using the following gasmixtures.

                  TABLE III    ______________________________________    GAS CONCENTRATIONS (%)    Gas      Level a       Level b Level c    ______________________________________    CO.sub.2 6.10          3.15    1.77    O.sub.2  5.42          11.60   17.45    N.sub.2  88.48         85.25   80.78    ______________________________________

After tonometry equilibrium was established, the pH, pO₂, and pCO₂ ofthe solutions remained constant even after a prolonged period oftonometry. Aliquots of the tonometered solutions were then transferredinto glass ampoules while purging with the same gas mixture used in thesolution tonometry. The filled ampoules were then flame-sealed.

The three levels of quality control standard having the combinationslisted in TABLES I, II, and III were then evaluated by two types ofinstruments: ANALYZER 1=Ciba Corning M288 blood gas analyzer andANALYZER 2=Ciba Corning M270 CO-oximeter. Results are summarized inTABLE IV.

                  TABLE IV    ______________________________________    Evaluations of Reference Standards    ANALYTE      LEVEL a   LEVEL b    LEVEL c    ______________________________________    ANALYZER 1    (Blood Gas)    pH           7.15 ± 0.02                           7.4 ± 0.02                                      7.6 ± 0.02    pCO.sub.2  mm Hg                 70 ± 2 40 ± 2  21 ± 2    pO.sub.2  mm Hg                 63 ± 2 100 ± 3 150 ± 5    Na mM        115 ± 2                           135 ± 2 155 ± 3    K mM         2.9 ± 0.3                           4.8 ± 0.3                                      7.1 ± 0.3    Cl mM        120 ± 2                           100 ± 2 80 ± 2    iCa mM       1.6 ± 0.05                           1.15 ± 0.05                                      0.65 ± 0.05    ANALYZER 2    (CO-oximeter)    THb g/dL     8.0 ± 0.2                           14 ± 0.2                                      18 ± 0.2    O.sub.2 Hb % 78 ± 2 95 ± 3  60 ± 2    COHb %       3.5 ± 1                           2.5 ± 1 6 ± 1    MetHb %      16 ± 2 1 ± 0.3 18 ± 2    HHb %        2.5 ± 1                           1.5 ± 0.7                                      16 ± 2    ______________________________________

FIG. 4 provides the absorbance spectra of the three dye-based qualitycontrol standard described above, where spectrum a represents level a;spectrum b represents a level b; and spectrum c represents level c, eachof which were measured using a 0.1 mm cuvette.

Example 2

A three level quality control standard was prepared by the procedure ofExample 1. To the bulk solutions described in TABLE I of Example 1 thedye formulations shown in TABLE V below were added.

                  TABLE V    ______________________________________    DYE FORMULATIONS    Dye           Level a   Level b   Level c    ______________________________________    Sulforhodamine B                  1.27 mM   2.53 mM   3.22 mM    Tartrazine    1.34      1.87      1.60    Patent Blue Violet                  0.11      0.17      0.25    Naphthol Green B                  0.98      1.96      1.64    ______________________________________

The formulated solutions were then tonometered at 25° C. to equilibriumusing the same gas mixtures described in TABLE III of Example 1. Thetonometered solutions were then transferred into glass ampoules whilepurging the same gas mixture used in the solution tonometry. The filledampoules were flame-sealed. Those three levels of reference weremeasured in the same manner as described in Example 1. The measuredranges were similar to the data shown in Example 1.

FIG. 5 provides the absorbance spectra of the three level dye-basedquality control standard described above, where spectrum a representslevel a; spectrum b represents a level b; and spectrum c representslevel c, each of which were measured using a 0.1 mm cuvette.

Example 3

A three level quality control standard was prepared by the sameprocedure of Example 1. The bulk solutions of TABLE I were combined withthe dye formulations of TABLE VII below.

                  TABLE VII    ______________________________________    Dye Formulations    Dye           Level a   Level b   Level c    ______________________________________    Sulforhodamine B                  0.93 mM   2.14 mM   2.37 mM    Mordant Yellow 10                  8.95      9.24      4.91    patent blue VF                  0.12      0.13      0.08    Naphthol Green B                  --        1.33      0.81    ______________________________________

The formulated solutions were tonometered and transferred into ampoules,sealed, and thereafter measured, as described in Example 1. The dataobtained using this formulation were similar to those measured inExample 1.

FIG. 6 provides the absorbance spectra of the three level dye-basedquality control standard described above, where spectrum a representslevel a; spectrum b represents a level b; and spectrum c representslevel c, each of which were measured using a 0.1 mm cuvette.

Example 4

A three level quality control standard was prepared by the sameprocedure of Example 1. The bulk solutions of TABLE I were combined withthe dye formulations of TABLE VIII below.

                  TABLE VIII    ______________________________________    Dye Formulations    Dye           Level a   Level b   Level c    ______________________________________    Sulforhodamine B                  1.17 mM   1.97 mM   2.19 mM    Mordant Yellow 7                  5.33      6.14      2.62    FD&C Blue 1   0.03      0.04      0.03    Naphthol Green B                  0.97      2.30      0.14    ______________________________________

The formulated solutions were tonometered and transferred into ampoules,sealed, and thereafter measured, as described in Example 1. The dataobtained using this formulation were similar to those measured inExample 1.

FIG. 7 provides the absorbance spectra of the three level dye-basedquality control standard described above, where spectrum a representslevel a; spectrum b represents a level b; and spectrum c representslevel c, each of which were measured using a 0.1 mm cuvette.

That which is claimed is:
 1. A method for preparing a reference materialfor CO-oximeter instrument systems comprising the steps of:a) selectinga red dye having a main absorption maxima at a wavelength region betweenabout 560 to about 580 nm to provide a basic spectrum, said red dyeselected from the group consisting of sulforhodamine B, chlorophenolred, xylenol orange, sulforhodamine 101 and combinations thereof; b)modifying said main absorption maxima of said red dye by adding a seconddye having an absorption maxima at a wavelength region between about 350to about 450 nm, said second dye selected from the group consisting ofmordant yellow 7, tartrazine, orange G, hydroxypyrenetrisulfonic acidmordant yellow 10 and combinations thereof; and c) establishing a newrelative baseline of said spectrum at wavelengths greater than about 640nm by adding a third dye to create a sufficient absorbance at about 630nm to give positive CO-ox fraction values for oxyhemoglobin,carboxyhemoglobin, methemoglobin and reduced hemoglobin when measured bysaid CO-oximeter instrument systems, said third dye selected from thegroup consisting of brilliant blue FC, erioglaucine, lissamine,alphazurine A, patent blue violet, hydroxy naphthol blue, patent blueVF, IR 125, naphthol green B and combinations thereof; wherein when saidred dye is selected from the group consisting of sulforhodamine B,chlorophenol red, xylenol orange, sulforhodamine 101 and combinationsthereof, said second dye is selected from the group consisting ofmordant yellow 7, orange G, hydroxypyrenetrisulfonic acid mordant yellow10 and combinations thereof and said third dye is selected from thegroup consisting of brilliant blue FC, erioglaucine, lissamine,alphazurine A, patent blue violet, hydroxy naphthol blue, patent blueVF, IR 125, naphthol green B and combinations thereof, or said seconddye is selected from the group consisting of mordant yellow 7,tartrazine, orange G, hydroxypyrenetrisulfonic acid mordant yellow 10and combinations thereof and said third dye is selected from the groupconsisting of brilliant blue FC, lissamine, alphazurine A, patent blueviolet, hydroxy naphthol blue, patent blue VF, IR 125, naphthol green Band combinations thereof; or wherein when said red dye is selected fromthe group consisting of chlorophenol red, xylenol orange, sulforhodamine101 and combinations thereof, said second dye is selected from the groupconsisting of mordant yellow 7, tartrazine, orange G,hydroxypyrenetrisulfonic acid mordant yellow 10 and combinations thereofand said third dye is selected from the group consisting of brilliantblue FC, erioglaucine, lissamine, alphazurine A, patent blue violet,hydroxy naphthol blue, patent blue VF, IR 125, naphthol green B andcombinations thereof.
 2. A method according to claim 1 wherein said reddye has an absorption band width of less than about 50 nm and anabsorption of near about zero at a wavelength of greater than about 610nm.
 3. A method according to claim 1 wherein said second dye shifts saidmain absorption maxima to a wavelength in the region of about 570 toabout 580 nm.
 4. A method according to claim 1 wherein said third dye isa combination of two dyes, one of said two dyes is a blue dye having anabsorption peak at about 625 to about 640 nm selected from the groupconsisting of brilliant blue FC, erioglaucine, lissamine, alphazurine A,patent blue violet, hydroxy naphthol blue, and patent blue VF, and thesecond of said two dyes is a dye with an absorbance maxima positioned ata wavelength of from about 700 to about 780 nm and an absorption bandwidth of from about 150 to about 200 nm selected from the groupconsisting of IR 125 and napthol green B.
 5. A method according to claim4 wherein said reference provides normal physiological CO-ox fractionsof: oxyhemoglobin 94-98%, carboxyhemoglobin 0 about 1% methemoglobin 0to about 1.5%, and reduced hemoglobin from about 1 to about 5%.
 6. Areference material comprising:(a) a first dye having a main absorptionmaxima at a wavelength between about 560 to about 580 nm, said first dyeselected from the group consisting of sulforhodamine B, chlorophenolred, xylenol orange, sulforhodamine 101 and combinations thereof; (b) asecond dye having an absorption maxima at a wavelength between about 350to about 450 nm, said second dye selected from the group consisting ofmordant yellow 7, tartrazine, orange G, hydroxypyrenetrisulfonic acid,mordant yellow 10, and combinations thereof; and (c) a third dye havingan absorption maxima at a wavelength between about 625 and about 640 nm,said third dye selected from the group consisting of selected from thegroup consisting of brilliant blue FC, erioglaucine, lissamine,alphazurine A, patent blue violet, hydroxy napthol blue, patent blue VFand combinations thereof; and (d) a fourth dye having an absorptionmaxima at a wavelength between about 700 and 780 nm and an absorptionband width of from about 150 to about 200 nm, said fourth dye selectedfrom the group consisting of IR 125, napthol green B and combinationsthereof, each of said dyes present in a sufficient amount to providesaid material with a sufficient absorbance at about 630 nm to provideclinically meaningful positive CO-ox fraction values of oxyhemoglobin,carboxyhemoglobin, methemoglobin and reduced hemoglobin.
 7. A materialaccording to claim 6 wherein each of said dyes present in a sufficientamount to provide said material with clinically meaningful CO-oxfractions.
 8. A method for quality control of CO-oximeter instrumentsystems comprising:1) subjecting said instrument to a quality controlstandard material, said material prepared according to a methodcomprising the steps of:a) selecting a red dye having a main absorptionmaxima at a wavelength region between about 560 to about 580 nm toprovide a basic spectrum, said red dye selected from the groupconsisting of sulforhodamine B, chlorophenol red, xylenol orange,sulforhodamine 101 and combinations thereof; b) modifying said mainabsorption maxima of said red dye by adding a second dye having anabsorption maxima at a wavelength region between about 350 to about 450nm, said second dye selected from the group consisting of mordant yellow7, tartrazine, orange G, hydroxypyrenetrisulfonic acid mordant yellow 10and combinations thereof; and c) establishing a new relative baseline ofsaid spectrum at wavelengths greater than about 640 nm by adding a thirddye to create a sufficient absorbance at about 630 nm to give positiveCO-ox fraction values for oxyhemoglobin, carboxyhemoglobin,methemoglobin and reduced hemoglobin when measured by said CO-oximeterinstrument systems, said third dye selected from the group consisting ofbrilliant blue FC, erioglaucine, lissamine, alphazurine A, patent blueviolet, hydroxy naphthol blue, patent blue VF, IR 125, naphthol green Band combinations thereof, said dyes present in an amount sufficient toprovide said control with a predetermined level of CO-ox fractions; 2)obtaining and instrument measurement of CO-ox fraction of said control;and 3) comparing said instrument measurement of said CO-ox fractions ofsaid control with said predetermined level of CO-ox fractions to checkthe accuracy of said instrument; wherein when said red dye is selectedfrom the group consisting of sulforhodamine B, chlorophenol red, xylenolorange, sulforhodamine 101 and combinations thereof, said second dye isselected from the group consisting of mordant yellow 7, orange G,hydroxypyrenetrisulfonic acid mordant yellow 10 and combinations thereofand said third dye is selected from the group consisting of brilliantblue FC, erioglaucine, lissamine, alphazurine A, patent blue violet,hydroxy naphthol blue, patent blue VF, IR 125, naphthol green B andcombinations thereof, or said second dye is selected from the groupconsisting of mordant yellow 7, tartrazine, orange G,hydroxypyrenetrisulfonic acid mordant yellow 10 and combinations thereofand said third dye is selected from the group consisting of brilliantblue FC, lissamine, alphazurine A, patent blue violet, hydroxy naphtholblue, patent blue VF, IR 125, naphthol green B and combinations thereof;or wherein when said red dye is selected from the group consisting ofchlorophenol red, xylenol orange, sulforhodamine 101 and combinationsthereof, said second dye is selected from the group consisting ofmordant yellow 7, tartrazine, orange G, hydroxypyrenetrisulfonic acidmordant yellow 10 and combinations thereof and said third dye isselected from the group consisting of brilliant blue FC, erioglaucine,lissamine, alphazurine A, patent blue violet, hydroxy naphthol blue,patent blue VF, IR 125, naphthol green B and combinations thereof.
 9. Amethod for quality control of CO-oximeter instrument systemscomprising:(1) subjecting said instrument to a quality control standardmaterial comprising:(a) a first dye having a main absorption maxima at awavelength between about 560 to about 580 nm, said first dye selectedfrom the group consisting of sulforhodamine B, chlorophenol red, xylenolorange, sulforhodamine 101 and combinations thereof; (b) a second dyehaving an absorption maxima at a wavelength region between about 350 toabout 450 nm, said second dye selected from the group consisting ofmordant yellow 7, tartrazine, orange G, hydroxypyrenetrisulfonic acid,mordant yellow 10, and combinations thereof; (c) a third dye having anabsorption maxima at a wavelength between about 625 and about 640 nm,said third dye selected from the group consisting of selected from thegroup consisting of brilliant blue FC, erioglaucine, lissamine,alphazurine A, patent blue violet, hydroxy napthol blue, patent blue VFand combinations thereof; and (d) a fourth dye having an absorptionmaxima at a wavelength between 700 to about 780 nm and an absorptionbandwidth of from about 150 to about 200 nm said fourth dye selectedfrom the group consisting of IR 125, napthol green B and combinationsthereof, wherein said dyes are used in a sufficient amount to providesaid control with a sufficient absorbance at about 630 nm to providepredetermined level of CO-ox fraction values of oxyhemoglobin,carboxyhemoglobin, methemoglobin and reduced hemoglobin; (2) obtainingan instrument measurement of CO-ox fractions of said control; and (3)comparing said instrument measurement of said CO-ox fractions of saidcontrol with said predetermined level of CO-ox fractions to check theaccuracy of said instrument.
 10. A method according to claim 9 whereinthree levels of quality control standards are prepared.